The internal elastic lamina (IEL) serves as a barrier for cells and macromolecules between the intima and media in the vascular wall. We evaluated the morphological changes and quantitative assessments of the IEL architecture in the coronary circulation of pigs fed with a high cholesterol diet. Transmission electron microscopy (TEM) analysis of the IEL from hypercholesterolemic coronary arteries revealed fragmentation of the IEL associated with a decrease in the thickness. Confocal microscopy and scanning electron microscopy (SEM) revealed an altered pattern characterized by a large oval fenestration in the IEL of hypercholesterolemic vessels. Morphometric analysis of confocal microscopy images demonstrated that the IEL of cholesterol-fed animals were characterized by an increase in the minor diameter of the fenestrae (2.16±0.04 µm vs 3.32±0.06 µm, p=0.003) and a decrease in the fenestrae density (22,333±1,334/mm² vs 17,552±931/mm², p=0.015) compared to controls. The percentage of the IEL area covered by the fenestrae correlated with the intimal thickness (r=0.79, p=0.004). The immunoreactivity for matrix metalloproteinase-3 (MMP-3) increased in cholesterol-fed coronary arteries, predominantly in the neointima. This study demonstrates experimental hypercholesterolemia induced ultrastructural changes of the IEL in the coronary circulation. The IEL may play an important role in the development of structural changes which characterize the early phase of coronary atherosclerosis.